Microfluidic differential extraction cartridge

ABSTRACT

A differential extraction system is directed to a microfluidic-based integrated cartridge to automate differential extraction of specific cell types within a mixed sample. The integrated cartridge includes a sonication module for selective cell lysis, separating means to eliminate centrifugation, high surface area pillar chip modules to purify DNA from a cell lysate, and microfluidic circuitry to integrate the steps in an automated platform.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No., ______, filed Sep. 17, 2004, and entitled “Sonication toSelectively Lyse Different Cell Types”, which claims priority under 35U.S.C. 119(e) of the co-pending U.S. Provisional Patent Application Ser.No. 60/504,072, filed Sep. 19, 2003, and entitled “MicrofluidicDifferential Extraction Instrument”, both of which are herebyincorporated by reference.

FIELD OF THE INVENTION

The invention relates to a method and apparatus for differential celllysis. In particular, the invention relates to differential cell lysisusing an integrated cartridge.

BACKGROUND OF THE INVENTION

Cell lysis is the destruction, or disruption, of a cell's membrane orwall, which breaks open the cell and exposes its contents. Manytechniques are available for the disruption of cells, including physicaland detergent-based methods. Physical lysis often requires expensive,cumbersome equipment and involves protocols that are difficult to repeatdue to variability in the apparatus. Detergent-based methods are ofteneasier-to-use with more efficient protocols than physical methods.

Sonication is one method of physically lysing cells. Sonication usespulsed, high frequency sound waves to agitate and lyse cells, bacteria,spores, and finely diced tissue. The sound waves are delivered using anapparatus with a vibrating probe that is immersed in the liquid cellsuspension. Mechanical energy from the probe initiates the formation ofmicroscopic vapor bubbles that form momentarily and implode, causingshock waves to radiate through the sample. The sonic energy delivered toa sample using this method is variable, and not repeatable for anecessary level of precision.

Cells can be treated with various agents to aid in the disruptionprocess. For example, lysis can be promoted by suspending cells in ahypotonic buffer, which cause them to swell and burst more readily underphysical shearing. Alternatively, processing can be expedited bytreating cells with glass beads in order to facilitate the crushing ofthe cell walls.

The resistance of cells and viruses to lysis or disruption is based onthe characteristics of the cell membrane, cell wall, or coat protein.The various chemical, enzymatic, and mechanical or physical approacheshave been utilized to non-specifically lyse cells and viruses. However,in some applications, it is desirable to lyse one specific cell type orvirus in a mixed sample of two or more cells and viruses. One suchapplication is DNA typing.

DNA typing has been an invaluable tool for forensic science.Applications including linking a suspect to a crime site or a victim,identifying a perpetrator via a “cold hit” in a networked crimelaboratory DNS database, identifying a victim or human remains, andproving the innocence of wrongly incarcerated prisoners by analyzingarchived evidence. Sample types and matrices can vary considerably, andthe entire sample preparation process can be very time consuming andlabor intensive.

Rape kits, containing swab samples of biological evidence collected inhospitals from victims of sexual assault, are amongst the most common,yet difficult, sample types to process since the swabs potentiallycontain a mixture of female epithelial cells and male sperm cells.Differential extraction is applied to separate the two distinct celltypes into male and female cell lysate fractions, and extract and purifythe DNA from each fraction. A resultant genetic profile of the male DNAis compared to that of a suspect, if available, or screened through thecrime laboratory DNA database.

The conventional method for separating epithelial cells from sperm cellsinvolves selective lysis using a combination of enzymes, chemicals,heat, and centrifugation. In a mixed sample, the epithelial cells arelysed first due to their lack of a protective coat, the sperm cells arepelleted using centrifugation, the epithelial cell lysate is removed,the sperm cells are re-suspended, and the sperm cells are lysed usingmore stringent enzymatic, chemical, and heat conditions. Thisconventional process takes hours, sometimes days. New cost-effective andefficient methods and instrumentation need to be developed and validatedfor practical low-cost, low, processing time, high-throughput solutions.

SUMMARY OF THE INVENTION

In an aspect of the present invention, a method selectively lyses aspecific cell type. The method comprises providing a sample including atleast two different cell types, lysing a first cell type to form a firstlysate while at least one remaining cell type remains intact, andseparating the first lysate from the sample. The first cell type can belysed at a first sonication energy and the lysing the first cell typecomprises applying the first sonication energy to the sample. Lysing thefirst cell type can comprise applying a first chemical treatment to thesample. The method can also include lysing a second cell type to form asecond lysate. Lysing the second cell type can comprise applying asecond chemical treatment to the sample. The second cell type can belysed at a second sonication energy and lysing the second cell typecomprises applying the second sonication energy to the sample.

In another aspect of the present invention, an integrated cartridgeperforms differential lysis. The integrated cartridge comprises a sampleinput chamber to receive a sample, the sample having at least twodifferent cell types, means for selectively lysing a first cell typewhile leaving intact any remaining cell types within the sample, therebyforming a first lysate, separating means to separate the first lysatefrom the sample, a collection vessel to collect the first lysate, andmicrofluidic circuitry to couple the sample input chamber, theseparating means, and the collection vessel. The sample input chambercan comprise a sonication chamber and the means for selectively lysingcomprises means for applying a first sonication energy within thesonication chamber, the first sonication energy sufficient to lyse thefirst cell type. The integrated cartridge can also include means forapplying an additive to the sonication chamber prior to applying thefirst sonication energy. The additive can be glass beads, chemicals or acombination thereof. The integrated cartridge can also include means forapplying a second sonication energy within the sonication chamber tolyse a second cell type within the sample after the first lysate isseparated from the sample, the second sonication energy sufficient tolyse the second cell type. The integrated cartridge can also includemeans for applying a chemical treatment to the sample after the firstlysate is separated from the sample, wherein the chemical treatmentlyses a second cell type. The integrated cartridge can be automated. Themeans for selectively lysing the first cell type can comprise means forapplying a chemical treatment to the sample. The collection vessel caninclude a mixing chamber to add and mix a solution to the first lysate.The integrated cartridge can also include a first purification chipcoupled to the mixing chamber to purify and concentrate a nucleic acidfrom the first lysate. The integrated cartridge can also include asecond collection vessel to collect a remaining sample after the firstlysate is separated from the sample. The second collection vessel caninclude a second mixing chamber to add and mix a solution to theremaining sample. The second mixing chamber can include means for lysinga second cell type within the remaining sample to form a second lysate.The means for lysing the second cell type can include means for applyinga chemical treatment, means for applying a heat treatment, or acombination thereof. The integrated cartridge can also include a secondpurification chip coupled to the second mixing chamber to purify andconcentrate a nucleic acid from the second lysate. The integratedcartridge can also include a heat plate removably coupled to the secondmixing chamber, the second purification chip, or a combination thereof.The integrated cartridge can also include means for adding a solution tothe sample chamber, the collection vessel, and the second collectionvessel. The means for adding a solution can be a mounting plateremovably coupled to the sample chamber, the collection vessel, and thesecond collection vessel. The means for selectively lysing the firstcell type can include a means for applying a chemical treatment to thesample within the sample chamber.

In yet another aspect of the present invention, an integrated cartridgeperforms differential lysis of female epithelial cells and male spermcells. The integrated cartridge comprises a sample input chamber toreceive a sample, the sample including the female epithelial cells andthe male sperm cells, means for selectively lysing the female epithelialcell while leaving intact the male sperm cells within the sample,thereby forming an epithelial cell lysate, separating means to separatethe epithelial cell lysate from the sample, a first mixing chamber tocollect the epithelial cell lysate, and microfluidic circuitry to couplethe sample input chamber, the separating means, and the first mixingchamber. The first mixing chamber can include at least two connectedchambers. The sample input chamber can comprise a sonication chamber andthe means for selectively lysing comprises means for applying a firstsonication energy within the sonication chamber, the first sonicationenergy sufficient to lyse the female epithelial cells. The integratedcartridge can also include means for applying an additive to thesonication chamber prior to applying the first sonication energy. Theadditive can be glass beads, chemicals, or a combination thereof. Theintegrated cartridge can also include means for applying a secondsonication energy within the sonication chamber to lyse the male spermcells within the sample after the epithelial cell lysate is separatedfrom the sample, the second sonication energy sufficient to lyse themale sperm cells. The integrated cartridge can also include means forapplying a chemical treatment to the sample after the epithelial celllysate is separated from the sample, wherein the chemical treatmentlyses male sperm cells. The integrated cartridge can be automated. Themeans for selectively lysing the female epithelial cells can comprisemeans for applying a chemical treatment to the sample. The collectionvessel can include a mixing chamber to add and mix a solution to theepithelial cell lysate. The integrated cartridge can also include afirst purification chip coupled to the mixing chamber to purify andconcentrate a nucleic acid from the epithelial cell lysate. Theintegrated cartridge can also include a second collection vessel tocollect a remaining sample after the epithelial cell lysate is separatedfrom the sample. The second collection vessel can include a secondmixing chamber to add and mix a solution to the remaining sample. Thesecond mixing chamber can include means for lysing male sperm cellswithin the remaining sample to forma sperm cell lysate. The means forlysing the male sperm cells can include means for applying a chemicaltreatment, means for applying a heat treatment, or a combinationthereof. The integrated cartridge can also include a second purificationchip coupled to the second mixing chamber to purify and concentrate anucleic acid from the sperm cell lysate. The integrated cartridge canalso include a heat plate removably coupled to the second mixingchamber, the second purification chip, or a combination thereof. Theintegrated cartridge can also include means for adding a solution to thesample chamber, the collection vessel, and the second collection vessel.The means for adding a solution can be a mounting plate removablycoupled to the sample chamber, the collection vessel, and the secondcollection vessel. The means for selectively lysing the first cell typecan include a means for applying a chemical treatment to the samplewithin the sample chamber.

In still yet another aspect of the present invention, an integratedcartridge performs differential lysis. The integrated cartridgecomprises a sample input chamber to receive a sample, the sample havinga first cell type, means for selectively lysing the first cell type,thereby forming a first lysate, a purification chip to purify andconcentrate a nucleic acid from the first lysate, and microfluidiccircuitry to couple the sample input chamber, the means for selectivelylysing, and the purification chip.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a conceptual block diagram of a sonication apparatusto selectively lyse a mixed sample using sonication according to a firstembodiment of the present invention.

FIG. 2 illustrates a conceptual block diagram of a sonication apparatusto selectively lyse a mixed sample using sonication according to asecond embodiment of the present invention.

FIG. 3 illustrates a conceptual block diagram of a sonication apparatusto selectively lyse a mixed sample using sonication according to a thirdembodiment of the present invention.

FIG. 4 illustrates a general method of performing selective lysis andprotein purification.

FIG. 5 illustrates an exemplary method in which a sonication apparatusof the present invention is applied to a rape kit.

FIG. 6 illustrates an integrated cartridge according to the preferredembodiment of the present invention.

FIG. 7 illustrates the integrated cartridge coupled to a mounting plate.

FIG. 8 illustrates the integrated cartridge coupled to a heating plate.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

Embodiments of a differential extraction system of the present inventionare directed to a microfluidic-based system to automate differentialextraction of specific cell types within a mixed sample. Differentialextraction is accomplished using different lysing protocols, such assonication, chemical, and heat. The microfluidic-based system includes amodule for selective cell lysis, separating means to eliminatecentrifugation, high surface area pillar chip modules to purify DNA froma cell lysate, and microfluidic circuitry to integrate the steps in anautomated platform.

In an embodiment, the differential extraction system is a sonicationapparatus which includes at least one sonication module for selectivelysis. The sonication module can include one or more sonicationchambers. In this embodiment, the sonication module includes twosonication chambers. Alternatively, one sonication chamber can be used.Still alternatively, three or more sonication chambers can be used whena mixed sample includes three or more different cell types. Each ofthese sonication module configurations is discussed in greater detailbelow.

Conventional sonication is an established physical method for rapid andnon-specific lysing of cells and viruses. As applied in the presentinvention sonication is used to selectively lyse cells and viruses.Sonication amplitude duration, and frequency can be adjusted to lysecertain cell types or viruses without lysing other cell types or virusesin a mixed sample. In addition, sonication can be used in combinationwith chemical and enzymatic approaches to further achieve higher cell orvirus lysis specificity. Other additives such as glass beads can also becombined with the sonication and/or chemical and enzymatic approaches.

In one exemplary application, forensic rape kit samples typicallyinclude sperm cells and female epithelial cells. In forensicapplications, embodiments of the present invention provide an automated,microfluidic system for differential extraction of samples having amixture of female epithelial cells and male sperm cells. The sonicationapparatus includes a sonication module for selective cell lysis and asilicon chip module for rapid DNA extraction, purification, andconcentration. This sonication apparatus is a turn-key system in whichan operator loads samples, starts the apparatus, and allows thesonication apparatus to run unattended while performing the entiredifferential extraction method. In an alternative embodiment, the sampleloading process is also automated.

In conventional methods, a mild 2 hour enzymatic/chemical step is firstused to lyse the softer epithelial cells. The sperm cells are pelletedby centrifugation and the epithelial cell lysate is removed. The spermcells are re-suspended and subjected to a strong 2 hourenzymatic/chemical step to lyse the tougher sperm cells. Using thesonication apparatus of the present invention, a few minutes of lowamplitude sonication replaces the mild 2 hour enzymatic/chemical stepand a few minutes of high amplitude sonication replaces the strongenzymatic/chemical step. Alternative separating means can also be usedin place of the centrifugation to separate the epithelial cell lysatefrom the intact sperm cells.

The present invention provides many advantages over conventional lysingmethods. The method of the present invention is much faster thanchemical and enzymatic approaches, the method is more repeatable, themethod provides improved cell or virus lysis selectively, and the methodis amenable to automation using robotics and/or microfluidics. In thepreferred embodiment, sonication chambers and separating means areembedded in a microfluidic system.

In the embodiments using sonication, each sonication chamber is of thetype described in U.S. Pat. No. 6,100,084. The sonication chamberincludes a container with a cavity therein for retaining a sample in anultrasonic transmission medium. The cavity is closed using a membrane.An electrode and a piezoelectric material are attached to the membrane,and a voltage source is electrically connected to the electrode and thepiezoelectric material. An amount of sonication energy necessary to lysea specific cell type is a function of many factors including, but notlimited to, the amount of voltage applied to the membrane, the thicknessof the membrane, the dimensions of the container and the cavity, thematerial of the container, the material of the membrane, a compositionof the transmission medium within the cavity, a duration of the appliedvoltage, and the type of cell to be lysed. As such, the sonicationenergy required to lyse a specific cell type is experimentallydetermined and in general is a relative function of the equipment used.As used herein, the term “sonication energy” is used to define an energyamplitude, frequency, duration, or any combination thereof. Use of thesonication chamber provides the ability to regulate the applied sonicenergy with high precision.

The microfluidic system includes microfluidic circuitry to process smallliquid volumes for complex reagent metering, mixing, and biochemicalanalysis. The microfluidic system provides a closed-loop environmentwhich minimizes environmental contamination and the potential ofcompromising the integrity of the sample.

FIG. 1 illustrates a conceptual block diagram of a sonication apparatus2 to selectively lyse a mixed sample using sonication according to afirst embodiment of the present invention. The mixed sample includes atleast two different cell types. For example, the sample can be a rapekit sample that includes female epithelial cells and male sperm cells. Afirst cell type within the mixed sample is lysed at a first sonicationenergy, and a second cell type is lysed at a second sonication energy.The apparatus 2 includes a first sonication chamber 10, a secondsonication chamber 20, a separating means 30, a first purification chip40, a second purification chip 50, a first output vessel 60, and asecond output vessel 70, each of which is coupled using microfluidiccircuitry, which is discussed in greater detail below. The mixed sampleis placed in the first sonication chamber 10. The first sonicationchamber 10 provides the first sonication energy to the mixed sample,thereby lysing the first cell type. The first sonication energy used tolyse the first cell type is lower than the second sonication energynecessary to lyse the second cell type. As a result, after the firstsonication energy is applied to the sample, the sample includes thefirst cell type lysate, referred to as a first lysate, and the secondcell type which is still intact. The separating means 30 separates thefirst lysate from the mixed sample. The first lysate passes through theseparating means 30 and is directed to the first purification chip 40.The separating means 30 preferably includes pores or openings that eachhave a diameter that is less than the diameter of the intact second celltype. Separation of a lysate from a sample is performed using anyconventional separating means. Examples of such separating meansinclude, but are not limited to, a filter, a membrane, magnetic beads, afrit, or any other means for separating a lysate from an intact celltype. The intact second cell type is blocked by the separating means 30and is directed to the second sonication chamber 20 by back flowing abuffer through the separating means 30.

The first purification chip 40 purifies and concentrates DNA from withinthe first lysate such that a first DNA is collected and the remainingportion of the first lysate passes through as waste. The collected firstDNA is collected in the first output vessel 60.

The second sonication chamber 20 provides the second sonication energyused to lyse the second cell type. The resulting second lysate isdirected to the second purification chip 50. The second purificationchip 50 purifies and concentrates DNA from within the second lysate suchthat a second DNA is collected and the remaining portion of the secondlysate passes through as waste. The collected second DNA is collected inthe second output vessel 70. Each of the purification chips 40 and 50are preferably of the type described in U.S. Pat. No. 5,707,799 and U.S.Pat. No. 5,952,173, which are both hereby incorporated by reference.

Although the sonication apparatus 2 is illustrated and described interms of selectively lysing two different cell types, it is understoodthat the mixed sample can include more than two different cell types andthat the sonication apparatus can selectively lyse more than two celltypes. In this case, the number of sonication chambers used isdetermined by the number of anticipated different cell types to belysed. Each cell type to be lysed must be lysed at a differentsonication energy than the other cell types within the mixed sample. Iftwo cell types are lysed at the same sonication energy, then it is notpossible to lyse one without lysing the other using this sonicationmethod. However, cell types that are uniquely lysed at a givensonication energy, can be selectively lysed using this sonicationmethod.

In an alternative embodiment, lysing of particular cell types can befacilitated by adding additives to the sample prior to any of thesonication steps. As with empirically determining the specificsonication energy required to lyse a particular cell type, the propertype and amount of additives to be used is also determinedexperimentally. Examples of additives include glass beads, chemicals,enzymes, or the addition of heat.

In still another alternative embodiment, the last remaining cell type inthe original mixed sample can be lysed using conventional chemicaland/or enzymatic lysing methods. For example, where a mixed sampleincludes two different cell types to be lysed, the first cell type islysed using the aforementioned sonication method at the first sonicationenergy, and the second cell type is lysed using a chemical/enzymaticlysing method. As another example, where a mixed sample includes threedifferent cell types, two of the cell types can be selectively lysed asdescribed above in relation to the sonication apparatus 2. The thirdcell type can be lysed using any conventional lysing method, includingsonication, chemical, or enzymatic.

Multiple sonication apparatuses 2 can also be coupled together inparallel. In such a configuration, multiple mixed samples can beprocessed in parallel to increase throughput.

It is also understood that various alternative combinations ofsonication chambers, separating means, and purification chips can beused to selectively lyse different cell types and to collect DNA fromthe resulting lysates. FIGS. 2 and 3 illustrate two such alternativeconfigurations.

FIG. 2 illustrates a conceptual block diagram of a sonication apparatus100 to selectively lyse a mixed sample using sonication according to asecond embodiment of the present invention. The sonication apparatus 100operates similarly to the sonication apparatus 2 except that the twosonication chambers 10 and 20 are replaced by a single sonicationchamber 110. In operation, the first sonication chamber 110 receives themixed sample and the first sonication energy is applied, thereby lysingthe first cell type and producing the first lysate. The first lysate isseparated from the mixed sample by the separating means 30, such thatthe first lysate passes through the separating means 30 and the intactsecond cell type is blocked by the separating means 30. The first lysateis directed to the first purification chip 40 as described above inrelation to the preferred sonication apparatus 2. The blocked secondcell type is back flushed from the separating means 30 to the sonicationchamber 110. The sonication chamber 110 applies the second sonicationenergy to the second cell type, thereby lysing the second cell type toproduce the second lysate. The second lysate is directed to the secondpurification chip 50 as described above in relation to the preferredsonication apparatus 2.

FIG. 3 illustrates a conceptual block diagram of a sonication apparatus200 to selectively lyse a mixed sample using sonication according to athird embodiment of the present invention. The sonication apparatus 200includes a single sonication chamber 210 and a single purification chip240. The sonication chamber 210 receives the mixed sample and providesthe first sonication energy as described in detail above. The firstlysate passes through the separating means 230 to the purification chip240. The purification chip 240 purifies and concentrates the first DNAfrom within the first lysate and the remaining portion of the firstlysate passes though as waste. Te collected first DNA is flushed fromthe purification chip 240 and is collected in the first output vessel260. Flushing the first DNA also acts to clean the purification chip240. Alternatively, after the first DNA is flushed from the purificationchip 240 to the first output vessel 260, a cleaning step is performed inwhich the purification chip 240 is rinsed with a cleaning solution toremove any remaining first lysate. The cleaning solution and anyaccompanying waste is removed from the sonication apparatus 200 usingthe microfluidic circuitry included therein.

The separating means 230 blocks the intact second cell type and theintact second cell type is collected in the sonication chamber 210. Thesecond sonication energy is provided by the sonication chamber 210,thereby lysing the second cell type. The second lysate passes throughthe separating means 230 into the purification chip 240. The second DNAis purified and collected within the purification chip 240 while theremaining portion of the second lysate passes through as waste. Thecollected second DNA is collected in a second output vessel 270.

It is understood that multiple sonication apparatuses 100 can be coupledtogether in parallel, and that multiple sonication apparatuses 200 canbe coupled together in parallel. Further, one or more sonicationapparatuses 2, one or more sonication apparatus 100, and/or one or moresonication apparatuses 200 can be coupled together in parallel tosimultaneously process multiple mixed samples.

Each sonication apparatus 2, 100, and 200 are configured as integratedmicrofluidic cartridges. An integrated cartridge avoids the necessity ofcomplicated, expensive robotic devices, while improving theeffectiveness and efficiency of fluid metering, mixing, and dispensing.The integrated cartridge can include a multilayer microfluidic plasticblock with fluidic circuitry and multiple independent active valves,syringe drives, a sonication lysis module including one or moresonication chambers as described above, one or more DNA purificationchips, reagent reservoirs, electronic hardware, and custom userinterface. Fluid flow through the integrated cartridge can bepressure-driven and computer-controlled. As such, each sonicationapparatus can be completely automated.

The integrated cartridge can include a liquid-handling module and apneumatic module. Each of these modules can be comprised of severallayers of machined polycarbonate plastic parts, which are sandwiched andsealed together using laser cut sheets of silicone gasket material. Thesilicone gasket material serves to seal the layers in each module andalso to function as diaphragm valves in the liquid-handling module. Tensto hundreds of independently controlled valves, working systematicallyto direct the flow of sample and reagents, can be created.

Syringe pumps can be used as a drive mechanism for moving, mixing,aspirating, and dispensing boluses of liquid between locations in theliquid-handling block. The syringe driver boards control a stepper motorthat moves the syringe plungers. Since the full stroke of the syringeshas 48,000 steps, high precision fluid metering can be accomplished. Forexample, the resolution for displacement of fluid using a 1 ml syringebarrel is 0.0208 ul per step. A variety of syringe sizes can beincorporated to accommodate fast, large volume movement and precisesmall volume metering.

Peristaltic pumps can also be used as the drive mechanism. Theperistaltic pump can achieve continuous flow and minimizes problems ofair in the lines.

The liquid-handling module can be mounted on the pneumatic module witho-rings around each pressure outlet to seal the pneumatic path. Valvesin the liquid-handling module are opened and closed by electricalactuation of three-way solenoid valves mounted on the backside of thepneumatic module. The liquid-handling module preferably include inletand outlet ports, microchannels, mixing chambers, at least onesonication chamber, and at least one DNA purification chip. Solutionsand reagents can be included within the cassette, or they can beintroduced from lines connected to external bulk containers.

A simple and intuitive Graphical User Interface (GUI) offers control tothe system hardware components. The GUI enables a user to fully controlthe syringe drive motion and speed, and all the pneumatically actuatedmembrane valves. The GUI also enables the user to script, store, load,and run protocols, which can be edited and modified throughout thecourse of protocol optimization.

The integrated cartridge can be configured to process multiple samplessimultaneously. In one embodiment, the cartridge can process 10 samplessimultaneously per run. If one run takes 30-45 minutes, then up to 480samples can be processed in a 24 hour period. The automation process canbe extended to use an automated feeder of samples to the microfluidiccassette.

The integrated cartridge can also include a DNA purification module. TheDNA purification module uses a DNA purification chip. The purificationchip is embedded in the plastic integrated cartridge. The purificationchip is a micromachined silicon structure having micropillars. Themicropillars create a high surface area within a collection chamber.Nucleic acids are captured on the pillars, washed, and released in asmall elution volume using standard chaotropic salt chemistry.Advantages to this flow through process include amenability to a systemintegration in a microfluidic platform and rapid extraction of nucleicacids from large volume samples. In addition, the high concentrationeffect permits using less sample and consequently less PCR (PolymeraseChain Reaction) reaction mix for PCR amplification.

Another advantage of the micropillar chip for DNA purification andconcentration is the ability to produce compact arrays of these chipsfor high throughput purification. An array of purification chips can beharbored in a microfluidic circuit. Each chip can have a dedicated lineand essentially be isolated form one another. Such a configuration canbe accomplished using microfluidics in which an intricate network ofchannels is confined to a relatively small area. Such an array can havebroad applications for preparing DNA from a wide variety of sampletypes. An exemplary array of DNA purification chips includes an 8×12array of 96 micropillar chips.

FIG. 4 illustrates a general method of performing selective lysis andprotein purification. At a step 400, a mixed sample is provided to asonication chamber. The mixed sample has two or more different celltypes, each cell type is lysed at a different sonication energy. Forexample, a first cell type lyses at an associated first sonicationenergy, a second cell type lyses at an associated second sonicationenergy, and so on. At a step 410, a buffer solution is added to themixed sample within the sonication chamber. At a step 420, a firstsonication energy is applied to the mixed sample, thereby lysing a firstcell type within the mixed sample. After sonication, the mixed samplecomprises a first lysate, which is the lysed first cell type, and aremaining portion of the mixed sample. The remaining portion of themixed sample includes intact cells for each cell type that has asonication energy greater than the first sonication energy alreadyapplied.

At a step 430, the first lysate is separated from the mixed sample. Aseparating means allows the first lysate to pass while blocking theremaining portion of the mixed sample. At a step 440, the first lysateis passed through a purification chip to purify and concentrate a firstprotein included within the first lysate. The first protein is collectedwithin the purification chip while a remaining portion of the firstlysate passes through the purification chip as waste. The first proteincollected in the purification chip is flushed and collected as a firstpurified protein sample.

Additional cell types in the remaining mixed sample can also be lysed.If a single remaining cell type is in the remaining mixed sample, thenthe remaining cell type can be lysed either using sonication or using aconventional lysing method such as a chemical and heat combination. Ifmore than one different cell type remain in the remaining samplemixture, than the steps 410 through 440 are repeated for each remainingcell type starting with the cell type with the lowest lysing sonicationenergy and working upward. Again, the last remaining cell type can belysed using sonication or other conventional lysing method.

At a step 450, a last remaining cell type within the remaining mixedsample is prepared for sonication. Where the last remaining cell type islysed using sonication, a buffer solution is added to the remainingmixed sample in preparation for sonication. Alternatively, the lastremaining cell type is leased using a conventional lysing method, thisalternative case, chemical additives can be added to the remaining mixedsample. At a step 460, the remaining cell type is leased to form a finallysate. At a step 470, the final lysate is passed through a purificationchip to purify and concentrate a final protein included within the finallysate. The final protein is collected within the purification chipwhile a remaining portion of the final lysate passes through thepurification chip as waste. The final protein collected in thepurification chip is preferably flushed and collected as a finalpurified protein sample.

FIG. 5 illustrates an exemplary method in which a sonication apparatusof the present invention is applied to a rape kit. In such anapplication, the rape kit includes a mixed forensic rape sample havingfemale epithelial cells and male sperm cells on a matrix. The matrix caninclude a swab or a swatch. It is desired to isolate the male DNA fromthe sperm cells, the female DNA from the epithelial cells, or both.According to a first method, two sonication steps are included. A firstsonication step uses a mild treatment to lyse epithelial cells but notsperm cells. A second sonication step uses a harsh treatment to lysesperm cells. In a second method, a chemical/heat treatment step isimplemented to lyse the sperm cells, thereby eliminating the requirementfor a second sonication chamber on the integrated platform.

In a step 500, the swab or swatch with the mixed rape sample is placedin the sonication chamber. At a step 510, a buffer solution is added tothe mixed rape sample. Preferably, the buffer solution is a 0.5-1 ml 10mM Tris-HCL with a pH of about 8.0. Alternatively, the buffer solutionis water. Still alternatively, the sonication chamber also includesglass beads. It is understood that the steps 500 and 510 can be reversedsuch that the sonication chamber already includes the buffer solutionprior to adding the mixed rape sample. At a step 520, a first sonicationenergy is applied to the mixed rape sample. As described above, thespecific amount of sonication energy necessary to lyse the epithelialcells is dependent on the specifications of the particular sonicationchamber used. Application of the first sonication energy releases boththe epithelial cells and the sperm cells from the matrix into the buffersolution. The first sonication energy is sufficient to selectively lysethe epithelial cells, however the more durable sperm cells remainintact. Sonication at the first sonication energy is considered a mildsonication. After application of the first sonication energy, the buffersolution includes the lysed epithelial cells and intact sperm cells.

At a step 530, the swab or swatch is preferably removed and the lysedepithelial cells are separated from the intact sperm cells. Theseparating means preferably includes a filter with pores or openingseach with a diameter of about 2 um. The sperm cells, which have about a5 um diameter, are blocked by the separating means, while the lysedepithelial cells pass through the separating means to a femalecollection vessel. In general, the diameter of the filter pores oropenings is large enough to allow the epithelial cell lysate to pass,but small enough to prevent the intact sperm cells from passing. Thesonication chamber is preferably rinsed with buffer to move theepithelial cell lysate through the separating means. At a step 540, thelysed epithelial cells are preferably mixed with an equal volume of bindsolution and passed through a purification chip where the femaleepithelial DNA is purified and concentrated from the remainingepithelial cell lysate. The remaining epithelial cell lysate passesthrough the purification chip as waste. The epithelial cell DNA iscollected from the purification chip.

At a step 550, the intact sperm cells are back flushed from theseparating means to a second sonication chamber and a 0.2 molar NaOH, orbasic solution, is added. Alternatively, the intact sperm cells are backflushed from the separating means to the sonication chamber where theepithelial cells were previously lysed. The NaOH solution preferablyincludes 50 mM DTT. Alternatively, proteinase K can also be added to thepreferred basic solution. At a step 560, a second sonication energy isapplied to lyse the sperm cells. The second sonication energy is greaterthan the first sonication energy used to lyse the epithelial cells.Sonication at the second sonication energy is considered a harshsonication. At a step 570, the lysed sperm cells are passed through theseparating means and directed a second purification chip. Alternatively,the lysed sperm cells can first be collected in a male collectionvessel, and later directed to the second purification chip. The lysedsperm cells are preferably mixed with an equal volume of bind solutionand passed through the second purification chip where the male sperm DNAis purified and concentrated from the remaining sperm cell lysate. Theremaining sperm cell lysate passes through the purification chip aswaste. The sperm DNA is collected from the purification chip.Alternatively, a single purification chip is used to collect both theepithelial DNA and the sperm DNA. In some applications, DNA purificationis not required and the collected intact sperm can be removed from thesonication apparatus prior to application of the second sonicationenergy.

In an alternative embodiment, the male sperm cells can be lysed bymethods other than sonication. For example, instead of performing thesteps 550 and 560, the intact sperm cells are collected in a malecollection vessel, basic solution is added, and the male collectionvessel is heated to a predetermined temperature for a predeterminedduration to lyse the male sperm cells.

In another alternative embodiment, after the first sonication, theintact sperm cells are separated from the epithelial cell lysate bysubjecting the mixture to centrifugation. The epithelial cell lysate isremoved from the sperm cell pellet. The sperm cell pellet isre-suspended and subjected to sonication at the second sonication energyto lyse the sperm cells. It should be understood that alternativeseparating means can be used, as described above.

As previously discussed, a systematic approach is taken to develop themost appropriate protocol for the differential extraction method. First,sonication threshold levels for sperm cells and epithelial cells aredetermined. It is known that sperm cells are more resistant tosonication than epithelial cells. DTT is known to break down thedisulfide bonds in the sperm coat. Sonication with the addition of DTTcan be used to facilitate sperm cell lysis. A typical procedure forspore lysis uses glass beads during sonication, and although the useglass beads during sonication does lyse sperm cells, glass beads arepreferably avoided for sperm cell lysis. Selective lysis of theepithelial cells is preferably accomplished using mild sonicationwithout DTT.

FIG. 6 illustrates an integrated cartridge 300 according to thepreferred embodiment of the present invention. The integrated cartridge300 is preferably configured to differentially lyse two different celltypes according to the methodology described above in relation to FIGS.4 and 5. In an exemplary application, the integrated cartridge 300 isused to differentially lyse female epithelial cells and male sperm cellsfrom a rape kit sample. The integrated cartridge 300 includes a sampleinput chamber 310 which also preferably functions as a sonicationchamber, a first set of mixing chambers 320, a second set of mixingchamber 330, a first purification chip 340, a second purification chip350, a first output vessel 360, a second output vessel 370, and a filter380. The female epithelial cells are lysed using sonication within thesonication chamber 310. In the preferred embodiment, a mounting seat 312is coupled to the outer bottom of the sonication chamber 310. Themounting seat 312 accepts a sonication horn and provides an interfacethrough which sonication energy is transmitted to the sonication chamber310. The mounting seat 312 is removably coupled to a sonication hornsuch that the sonication horn can be connected and disconnected from theintegrated cartridge 300. In this manner, the integrated cartridge 300can be coupled to any number of different sonication horns, or anynumber of integrated cartridges 300 can be sequentially coupled to asingle sonication born.

Solution including the lysed epithelial cells and the intact sperm cellsis directed to the filter 380 which passes the epithelial cell lysateand blocks the intact sperm cells. The epithelial cell lysate isdirected to the first set of mixing chambers 320. The first set ofmixing chambers 320 is used to mix the epithelial cell lysate with anydesired solution, such as a bind solution, in preparation for DNAconcentration and purification within the first purification chip 340.The first set of mixing chambers 320 preferably includes two independentchambers connected to each other. It is understood that more, or less,than two chambers can be used. The mixed solution from the first set ofmixing chambers 320 is directed through the first purification chip 340where the epithelial cell DNA is purified and concentrated while theremaining portion of the mixed solution passes through as waste. Theepithelial cell DNA is then washed into the first output vessel 360.

The intact sperm cells are preferably back flushed from the filter 380to the second set of mixing chambers 330. The second set of mixingchambers 330 is used to mix the intact sperm cells with any desiredsolution, such as the 0.2 molar NaOH solution. The second set of mixingchambers 330 preferably includes two independent chambers connected toeach other. It is understood that more, or less, than two chambers canbe used. Within the second set of mixing chambers, the intact spermcells are lysed. Lying sperm cells can be performed using chemicals,heat, or a combination thereof. In the preferred embodiment, theintegrated cartridge 300 is fitted to a heating plate 390 (FIG. 8) suchthat heat can be applied to all, or a portion of, the second set ofmixing chambers 330.

In an alternative embodiment, the intact sperm cells are back flushedfrom the filter 380 to the sonication chamber 310. In the sonicationchamber 310, the intact sperm cells are lysed using sonication.Chemicals and/or other additives, such as glass beads, can be added tothe sonication chamber 310 prior to application of the sonicationenergy. In this manner, the sperm cells can be lysed using sonication,or a combination of sonication and other additives.

Sperm cell lysate is then directed from either the sonication chamber310 or the second set of mixing chambers 330 to the second purificationchip 350. If the protocol demands, the sperm cell lysate can be mixedwith a desired solution within the second set of mixing chambers 330prior to passing through the second purification chip 350. Sperm cellDNA is purified and concentrated within the second purification chip350, while the remaining portion of the sperm cell lysate solutionpasses through as waste. The sperm cell DNA is then washed into thesecond output vessel 370.

FIG. 7 illustrates the integrated cartridge 300 coupled to a mountingplate 600. The mounting plate 600 preferably couples to the integratedcartridge 300 at select injection ports to provide mixing reagents andtransmission fluid for the microfluidic circuitry.

FIG. 8 illustrates the integrated cartridge 300 coupled to the heatingplate 390. The heating plate 390 includes heating elements to provideheat to a select portion of the second set of mixing chambers 330 (FIG.6), and to the second purification chip 350 (FIG. 6). Preferably, theheating elements are power resistors. As shown in FIG. 8, the heatingplate 390 includes three power resistors, each power resistor includestwo leads 392, 394, and 396. It is understood that more or less thanthree power resistors can used. It is also understood that heatingelements other than power resistors can be used. The heating plate 390is preferably fitted to the integrated cartridge 300 on the oppositeside of the mounting plate 600. The heating plate 390 and the mountingplate 600 are both removably fitted to the integrated cartridge 300 suchthat the integrated cartridge 300 can be easily removed.

Multiple mounting plates can be configured to concurrently acceptmultiple integrated cartridges. Each integrated cartridge 300 isconfigured to be connected and disconnected from a sonication horn, amounting plate, and a heating plate. Connection and disconnection can beperformed automatically within an automated system, or can be performedmanually.

In an alternative embodiment, an integrated cartridge includes asonication chamber, one set of mixing chambers, one purification chip,and one output vessel. In this alternative embodiment, the integratedcartridge is used to perform a single lysis step. An exemplaryapplication is to lyse a sample having only collected sperm cells.Protocols can use any combination of sonication, chemical, and heatsteps as described above. For example, sonication can be performedindependent of a chemical step and independent of a heat step. Or, achemical step and a heat step can be performed concurrently, andsonication can be used not for lysing but to remove the colleted celltype from the input sample matrix. Or, sonication can be performed on asample which has chemicals added to the solution.

It should be understood that the lysates can be purified using meansother than the described purification chips. For example, the epithelialand sperm cell lysates can be subjected to DNA purification using thepurification chip, glass membrane, glass column, organic extraction,ethanol precipitation, or any other method known to purify nucleicacids.

Although embodiments of the differential lysis apparatus of the presentinvention are described above as including at least a first sonicationchamber to lyse a first cell type using sonication, the differentiallysis apparatus can be generalized to perform differential lysis withoutthe use of sonication. As such, the differential lysis apparatus of thepresent invention, in general, ia an integrated cartridge that performsdifferential lysis on a given sample.

The present invention has been described in terms of specificembodiments incorporating details to facilitate the understanding of theprinciples of construction and operation of the invention. Suchreference herein to specific embodiments and details thereof is notintended to limit the scope of the claims appended hereto. It will beapparent to those skilled in the art that modifications may be made inthe embodiment chosen for illustration without departing from the spiritand scope of the invention.

1. A method of selectively lysing a specific cell type, the methodcomprising: a. providing a sample including at least two different celltypes; b. lysing a first cell type to form a first lysate while at leastone remaining cell type remains intact; and c. separating the firstlysate from the sample.
 2. The method of claim 1 wherein the first celltype is lysed at a first sonication energy and the lysing the first celltype comprises applying the first sonication energy to the sample. 3.The method of claim 1 wherein lysing the first cell type comprisesapplying a first chemical treatment to the sample.
 4. The method ofclaim 1 further comprising lysing a second cell type to form a secondlysate
 5. The method of claim 4 wherein lysing the second cell typecomprises applying a second chemical treatment to the sample.
 6. Themethod of claim 4 wherein the second cell type is lysed at a secondsonication energy and lysing the second cell type comprises applying thesecond sonication energy to the sample.
 7. An integrated cartridge toperform differential lysis, the integrated cartridge comprising: a. asample input chamber to receive a sample, the sample having at least twodifferent cell types; b. means for selectively lysing a first cell typewhile leaving intact any remaining cell types within the sample, therebyforming a first lysate; c. separating means to separate the first lysatefrom the sample; d. a collection vessel to collect the first lysate; ande. microfluidic circuitry to couple the sample input chamber, theseparating means, and the collection vessel.
 8. The integrated cartridgeof claim 7 wherein the sample input chamber comprises a sonicationchamber and the means for selectively lysing comprises means forapplying a first sonication energy within the sonication chamber, thefirst sonication energy sufficient to lyse the first cell type.
 9. Theintegrated cartridge of claim 8 further comprising means for applying anadditive to the sonication chamber prior to applying the firstsonication energy.
 10. The integrated cartridge of claim 9 wherein theadditive is glass beads, chemicals, or a combination thereof.
 11. Theintegrated cartridge of claim 8 further comprising means for applying asecond sonication energy within the sonication chamber to lyse a secondcell type within the sample after the first lysate is separated from thesample, the second sonication energy sufficient to lyse the second celltype.
 12. The integrated cartridge of claim 8 further comprising meansfor applying a chemical treatment to the sample after the first lysateis separated from the sample, wherein the chemical treatment lyses asecond cell tripe.
 13. The integrated cartridge of claim 7 wherein theintegrated cartridge is automated.
 14. The integrated cartridge of claim7 wherein the means for selectively lysing the first cell type comprisesmeans for applying a chemical treatment to the sample.
 15. Theintegrated cartridge of claim 7 wherein the collection vessel includes amixing chamber to add and mix a solution to the first lysate.
 16. Theintegrated cartridge of claim 15 further comprising a first purificationchip coupled to the mixing chamber to purify and concentrate a nucleicacid from the first lysate.
 17. The integrated cartridge of claim 7further comprising a second collection vessel to collect a remainingsample after the first lysate is separated from the sample.
 18. Theintegrated cartridge of claim 17 wherein the second collection vesselincludes a second mixing chamber to add and mix a solution to theremaining sample.
 19. The integrated cartridge of claim 18 wherein thesecond mixing chamber includes means for lysing a second cell typewithin the remaining sample to form a second lysate.
 20. The integratedcartridge of claim 19 wherein the means for lysing the second cell typeincludes means for applying a chemical treatment, means for applying aheat treatment, or a combination thereof.
 21. The integrated cartridgeof claim 20 further comprising a second purification chip coupled to thesecond mixing chamber to purify and concentrate a nucleic acid from thesecond lysate.
 22. The integrated cartridge of claim 21 furthercomprising a heat plate removably coupled to the second mixing chamber,the second purification chip, or a combination thereof.
 23. Theintegrated cartridge of claim 17 further comprising means for adding asolution to the sample chamber, the collection vessel, and the secondcollection vessel.
 24. The integrated cartridge of claim 23 wherein themeans for adding a solution is a mounting plate removably coupled to thesample chamber, the collection vessel, and the second collection vessel.25. The integrated cartridge of claim 7 wherein the means forselectively lysing the first cell type includes a means for applying achemical treatment to the sample within the sample chamber.
 26. Anintegrated cartridge to perform differential lysis of female epithelialcells and male sperm cells, the integrated cartridge comprising: a. asample input chamber to receive a sample, the sample including thefemale epithelial cells and the male sperm cells; b. means forselectively lysing the female epithelial cell while leaving intact themale sperm cells within the sample, thereby forming an epithelial celllysate; c. separating means to separate the epithelial cell lysate fromthe sample; d. a first mixing chamber to collect the epithelial celllysate; and e. microfluidic circuitry to couple the sample inputchamber, the separating means, and the first mixing chamber.
 27. Theintegrated cartridge of claim 26 wherein the first mixing chamberincludes at least two connected chambers.
 28. The integrated cartridgeof claim 26 wherein the sample input chamber comprises a sonicationchamber and the means for selectively lysing comprises means forapplying a first sonication energy within the sonication chamber, thefirst sonication energy sufficient to lyse the female epithelial cells.29. The integrated cartridge of claim 28 further comprising means forapplying an additive to the sonication chamber prior to applying thefirst sonication energy.
 30. The integrated cartridge of claim 29wherein the additive is glass beads, chemicals, or a combinationthereof.
 31. The integrated cartridge of claim 28 further comprisingmeans for applying a second sonication energy within the sonicationchamber to lyse the male sperm cells within the sample after theepithelial cell lysate is separated from the sample, the secondsonication energy sufficient to lyse the male sperm cells.
 32. Theintegrated cartridge of claim 28 further comprising means for applying achemical treatment to the sample after the epithelial cell lysate isseparated from the sample, wherein the chemical treatment lyses malesperm cells.
 33. The integrated cartridge of claim 26 wherein theintegrated cartridge is automated.
 34. The integrated cartridge of claim26 wherein the means for selectively lysing the female epithelial cellscomprises means for applying a chemical treatment to the sample.
 35. Theintegrated cartridge of claim 26 wherein the collection vessel includesa mixing chamber to add and mix a solution to the epithelial celllysate.
 36. The integrated cartridge of claim 35 further comprising afirst purification chip coupled to the mixing chamber to purify andconcentrate a nucleic acid from the epithelial cell lysate.
 37. Theintegrated cartridge of claim 26 further comprising a second collectionvessel to collect a remaining sample after the epithelial cell lysate isseparated from the sample.
 38. The integrated cartridge of claim 37wherein the second collection vessel includes a second mixing chamber toadd and mix a solution to the remaining sample.
 39. The integratedcartridge of claim 38 wherein the second mixing chamber includes meansfor lysing male sperm cells within the remaining sample to form a spermcell lysate.
 40. The integrated cartridge of claim 39 wherein the meansfor lysing the male sperm cells includes means for applying a chemicaltreatment, means for applying a heat treatment, or a combinationthereof.
 41. The integrated cartridge of claim 40 further comprising asecond purification chip coupled to the second mixing chamber to purifyand concentrate a nucleic acid from the sperm cell lysate.
 42. Theintegrated cartridge of claim 41 further comprising a heat plateremovably coupled to the second mixing chamber, the second purificationchip, or a combination thereof.
 43. The integrated cartridge of claim 38further comprising means for adding a solution to the sample chamber,the collection vessel, and the second collection vessel.
 44. Theintegrated cartridge of claim 43 wherein the means for adding a solutionis a mounting plate removably coupled to the sample chamber, thecollection vessel, and the second collection vessel.
 45. The integratedcartridge of claim 26 wherein the means for selectively lysing the firstcell type includes a means for applying a chemical treatment to thesample within the sample chamber.
 46. An integrated cartridge to performdifferential lysis, the integrated cartridge comprising: a. a sampleinput chamber to receive a sample, the sample having a first cell type;b. means for selectively lysing the first cell type, thereby forming afirst lysate; c. a purification chip to purify and concentrate a nucleicacid from the first lysate; and d. microfluidic circuitry to couple thesample input chamber, the means for selectively lysing, and thepurification chip.